The proprietary splitSENSOR technology is a highly sensitive, flexible and easy-to-use reporter assay system to monitor various cellular events in living cells, including the activity of drug targets. Various cellular events, such as regulated protein-protein interactions, can be robustly and quantitatively measured both at the membrane and in the cytosol of the cell. This feature makes it, for example, an invaluable tool to assess the activity of receptors as measured by the regulated binding to their cognate cytosolic adapters. Therefore, this technology can be applied in particular to assess compound actions on drug targets, such as G protein-coupled receptors and receptor tyrosine kinases.
As luciferase reporters are used as readout, the splitSENSOR technology is amenable to high-throughput applications including compound screening approaches. targetSCREENER assays, which are based on the splitSENSOR technology, are available for transient expression, and alternatively, assay components can be stably integrated into mammalian cells of choice using neomycin selection.
The power and applicability of the splitSENSOR technology was demonstrated by various publications in high-class scientific journals.
targetSCREENER assays allow the monitoring various types of cellular events, such as regulated protein-protein interactions, degradation events, translocation events, and cleavage events (clockwise starting from top).
How it works
The splitSENSOR technology is based on the functional reconstitution of two previously inactive fragments derived from the non-native NIa protease of the tobacco etch virus (TEV protease) (Wehr et al., 2006). These fragments, either an N-terminal (NTEV) or C-terminal part (CTEV) of the TEV protease, are fused to interaction partners of choice. Upon interaction of the two candidate proteins, the NTEV and CTEV fragments get into close proximity, thereby regaining proteolytic activity. An active protease recognises and cleaves its target sequence, ENLYFQG, to release a previously inactive reporter molecule, i.e. an artificial transcriptional co-activator like Gal4-VP16 (GV). GV, in turn, translocates to the nucleus to activate a reporter gene, such as firefly luciferase (Fluc, produces light).
The technology can be applied to monitor interactions at the membrane (left) and in the cytosol (right). For an efficient and robust assay involving membrane proteins or proteins that are localised to the sub-membrane compartment, the artificial transcriptional co-activator GV is fused to the NTEV moiety via a TEV protease cleavage site.